1. Field of the Invention
The present invention relates to novel cyclosporine derivatives that have improved protein and solid surface conjugatibility and hydrolytic stability. The cyclosporine derivatives of the present invention are useful in assays measurement of cyclosporin A levels, as well as in the production of cyclosporine immunogens and capture conjugates.
2. Background
Cyclosporine A (cyclosporine) is a potent immuno-suppressant that has been widely used in the United States and other countries to prevent the rejection of transplanted organs such as kidney, heart, bone marrow and liver, in humans.
To prevent allograft rejections, minimum level cyclosporine A in the blood is required throughout the lifetime of the patient. Chronic high doses can result in kidney and liver damage. Distribution and metabolism of the drug varies greatly between individuals, as well as in a single individual during the course of therapy. Accordingly, monitoring cyclosporine A levels in the blood or serum of allograph recipients is considered essential.
Laboratory methods for detection of cyclosporine have been developed. These techniques typically involve high performance liquid chromatography (HPLC), radioimmunoassay (RIA) and non-radioimmunoassay.
It has been reported that CsA, itself, is non-immunogenic (Donatsch, p. et al., J. Immuno Assay 1981; 2:19). To obtain antibodies, therefore, it is necessary to link CsA to a protein carrier. The side chain of CsA, however, consists most of alliphatic groups. Few of the functional groups customarily used to link a hapten to a carrier. Previous workers have made immunogenic cyclosporine C (CsC) protein conjugates because the CsC has a threonine residue in position 2. Linkage to a protein was via a hemisuccinate linker through an ester group (U.S. Pat. No. 5,169,773). In addition, hemisuccinate coupling chemistry has been used to immobilize CsC to a solid support such as stabilized chromium dioxide particles (U.S. Pat. No. 5,151,348). Due to a number of factors including, for example, short chain length of the hemisuccinate linker, hydrophobicity of the cyclosporine-C hemisuccinate molecule and hydrolytic instability of the hemisuccinate ester linkage, the CsC hemisuccinate derivatives conjugate poorly to protein and solid surface. Furthermore, CsC protein conjugates and CsC immobilized on a solid support by hemisuccinate coupling, are hydrolyticly unstable. Thus, immunoassays developed by using such hemisuccinate CsC derivatives suffer from low sensitivity and poor reagent stability. There is a strong desire to replace the widely used radioimmunoassays and HPLC methods with a more robust and sensitive immunoassay for CsA. Accordingly, there is a need in the art for cyclosporine derivatives that are capable of being conjugated to solid supports and carriers more efficiently and stably.